PROJECT SUMMARY Herpes Simplex Virus 1 (HSV-1) infection affects upwards of 50 percent of the population and poses an especial risk to infants and immunocompromised individuals. Identifying targets and understanding their roles during lytic infection could lead to the development of new therapeutics. Our laboratory used iPOND (Isolation of Proteins on Nascent DNA) to isolate the HSV-1 genome and associated viral and host cell proteins at different points in the HSV-1 lytic life cycle. The FACT (Facilitates Chromatin Transcription) complex was found to be associated with the HSV-1 genome both before and during viral DNA replication. The FACT complex is a heterodimer, consisting of the proteins Spt16 and SSRP1, and is involved in chromatin remodeling during transcription, DNA replication, recombination, and the DNA damage response. Preliminary data are provided showing that SSRP1 colocalized with the viral genome during viral DNA replication and that before viral DNA replication SSRP1 relocalized from the nucleolus into distinct puncta. Subsequent experiments showed that early relocalization required expression of the viral immediate early protein, ICP22, which was also found on infecting viral genomes by iPOND. We hypothesize that ICP22 alters the FACT complex's intranuclear location prior to viral DNA replication by direct interaction or through affecting other cellular proteins, and that ICP22 is involved in the association of the FACT complex with the HSV-1 genome during replication either directly or through affecting other cellular or viral proteins. We further hypothesize that the presence of the FACT complex decreases histone occupancy on the viral genome and supports processes that occur on the viral genome such as transcription. Three aims are proposed to explore how the FACT complex is recruited to HSV-1 DNA and how it might be involved in viral gene expression and replication. In Aim 1 we will define the mechanism by which SSRP1 relocalizes in the host cell nucleus prior to viral DNA replication. This includes determining if the association of the FACT complex with HSV-1 DNA prior to viral DNA replication is dependent on ICP22 and which viral and cellular proteins interact with the FACT complex prior to viral DNA replication. In Aim 2 we will explore the association of the FACT complex with the HSV- 1 genome during viral DNA replication and the role of ICP22. Immunoprecipiation experiments will allow us to determine which viral and cellular proteins interact with the FACT complex during viral DNA replication. In Aim 3 we will determine the function of the FACT complex during HSV-1 infection by observing the effects of a knockdown of Spt16 and SSRP1 on viral gene expression and replication. This will be correlated with changes in histone and transcription factor occupancy. This study will shed light on how this complex (FACT), which affects most DNA transactions, contributes to the life cycle of HSV-1.